Human antibodies to human delta like ligand 4

ABSTRACT

An isolated human antibody or a fragment of a human antibody which specifically binds to human delta-like ligand 4 (hDII4) and blocks hDII4 binding to a Notch receptor. The human anti-hDII4 antibody or antibody fragment binds dimeric hDII4 with an affinity of 75 pM or better, as measured by surface plasmon resonance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. non-provisional applicationSer. No. 12/002,245 filed Dec. 14, 2007, which claims the benefit under35 U.S.C § 119(e) of U.S. provisional application Nos. 60/874,922 filedDec. 14, 2006, 60/916,415 filed May 7, 2007, and 60/985,323 filed Nov.5, 2007, all of which are herein specifically incorporated by referencein their entirety.

BACKGROUND

The Notch-signaling pathway is a system for cell-to-cell communicationused by a wide range of eukaryotes for many biological processes, suchas differentiation, proliferation, and homeostasis. Delta like 4 (DI4)or delta-like ligand 4 (DII4) (hereinafter “DII4”) is a member of theDelta family of Notch ligands which exhibits highly selective expressionby vascular endothelium (Shutter et al. (2000) Genes Develop.14:1313-1318). DII4 is a ligand for Notch receptors, including Notch1and Notch 4. The nucleic acid and amino acid sequences for human DII4are shown in SEQ ID NO:1-2, respectively.

Methods to produce antibodies useful as human therapeutics includegeneration of chimeric antibodies and humanized antibodies (see, forexample, U.S. Pat. No. 6,949,245). See, for example, WO 94/02602(Abgenix) and U.S. Pat. No. 6,596,541 (Regeneron Pharmaceuticals), whichpublications are herein specifically incorporated by reference,describing methods of generating nonhuman transgenic mice capable ofproducing human antibodies.

Japanese patent application 2003/047470A2 (Asahi Kasei Kogyo) describesantibodies to the extracellular portion of human Notch ligand protein.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides human antibodies, preferablyrecombinant human antibodies, that specifically bind human delta-likeligand 4 (hDII4). These antibodies are characterized by binding to hDII4with high affinity and by the ability to neutralize DII4 activity. Theantibodies of the invention are capable of blocking DII4 binding to itsNotch receptor(s), and thus inhibit signaling by DII4. The antibodiescan be full-length (for example, an IgG1 or IgG4 antibody) or maycomprise only an antigen-binding portion (for example, a Fab, F(ab′)₂ orscFv fragment), and may be modified to effect functionality, e.g., toeliminate residual effector functions (Glu which eliminates residualeffector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933).

In one embodiment, the antibody of the invention comprises a heavy chainvariable region (HCVR) selected from the group consisting of SEQ IDNO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228,244, 260, 276, 292, 308, 324, 340, 356, 372, 397, 413, 429, 445, 461,477, 493, 509, 525, 541, 557, 573, 589, 605, 621, 637, 653, 669, 685,701, 717, 733, 749, 765, 781, 797, 813, 893, 897, 901, 905, 909, 913,917, 921, 925, 935, 939, 943, and 947 or a substantially identicalsequence thereof. In a preferred embodiment, the HCVR is the amino acidsequence of SEQ ID NO:429 or 901.

In one embodiment, the antibody of the invention comprises a light chainvariable region (LCVR) selected from the group consisting of SEQ IDNO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236,252, 268, 284, 300, 316, 332, 348, 364, 380, 405, 421, 437, 453, 469,485, 501, 517, 533, 549, 565, 581, 597, 613, 629, 645, 661, 677, 693,709, 725, 741, 757, 773, 789, 805, 821, 895, 899, 903, 907, 911, 915,919, 923, 927, 937, 941, 945, and 949 or a substantially identicalsequence thereof. In a preferred embodiment, the LCVR is the amino acidsequence of SEQ ID NO:437 or 903.

In one embodiment, the antibody of the invention comprises a HCVRselected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84,100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308,324, 340, 356, 372, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541,557, 573, 589, 605, 621, 637, 653, 669, 685, 701, 717, 733, 749, 765,781, 797, 813, 893, 897, 901, 905, 909, 913, 917, 921, 925, 935, 939,943, and 947 or a substantially identical sequence thereof, and a LCVRselected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92,108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316,332, 348, 364, 380, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549,565, 581, 597, 613, 629, 645, 661, 677, 693, 709, 725, 741, 757, 773,789, 805, 821, 895, 899, 903, 907, 911, 915, 919, 923, 927, 937, 941,945, and 949 or a substantially identical sequence thereof. In apreferred embodiment, the HCVR/LCVR are the amino acid sequence pairsSEQ ID NO:429/437 or 901/903.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain complementary determining region 1(CDR1) selected from the group consisting of SEQ ID NO:6, 22, 38, 54,70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278 294,310, 326, 342, 358, 374, 399, 415, 431, 447, 463, 479, 495, 511, 527,543, 559, 575, 591, 607, 623, 639, 655, 671, 687, 703, 719, 735, 751,767, 783, 799, 815, 831, 847, 863 and 879, or a substantially identicalsequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR2 selected from the groupconsisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168,184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 401,417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, 593, 609, 625,641, 657, 673, 689, 705, 721, 737, 753, 769, 785, 801, 817, 833, 849,865 and 881, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR3 selected from the groupconsisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170,186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 403,419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, 595, 611, 627,643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819, 835, 851,867 and 883, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR1 selected from the groupconsisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166,182, 198, 214, 230, 246, 262, 278 294, 310, 326, 342, 358, 374, 399,415, 431, 447, 463, 479, 495, 511, 527, 543, 559, 575, 591, 607, 623,639, 655, 671, 687, 703, 711119, 735, 751, 767, 783, 799, 815, 831, 847,863 and 879, or a substantially identical sequence thereof; a heavychain CDR2 selected from the group consisting of SEQ ID NO: 8, 24, 40,56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280,296, 312, 328, 344, 360, 376, 401, 417, 433, 449, 465, 481, 497, 513,529, 545, 561, 577, 593, 609, 625, 641, 657, 673, 689, 705, 721, 737,753, 769, 785, 801, 817, 833, 849, 865 and 881, or a substantiallyidentical sequence thereof; and a heavy chain CDR3 selected from thegroup consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138,154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362,378, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, 595,611, 627, 643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819,835, 851, 867 and 883, or a substantially identical sequence thereof. Ina preferred embodiment, the antibody or antibody fragment comprisesheavy chain CDR1, CDR2 and CDR3 selected from the group consisting ofSEQ ID NO:431/433/435; 374/376/378; 783/785/787; and 799/801/803.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR1 selected from the groupconsisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382,407, 423, 439, 455, 471, 487, 503, 519, 535, 551, 567, 583, 599, 615,631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839,855, 871 and 887, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR2 selected from the groupconsisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176,192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 409.425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, 601, 617, 633,649, 665, 681, 697, 713, 729, 745, 761, 777, 793, 809, 825, 841, 857,873 and 889, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR3 selected from the groupconsisting of SEQ ID NO:18, 34, 50, 66, 82, 98, 11, 130, 146, 162, 178,194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 411,427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, 603, 619, 635,651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827, 843, 859,875 and 891, or a substantially identical sequence thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR1 selected from the groupconsisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382,407, 423, 439, 455, 471, 487, 503, 519, 535, 551, 567, 583, 599, 615,631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839,855, 871 and 887, or a substantially identical sequence thereof; a lightchain CDR2 selected from the group consisting of SEQ ID NO: 16, 32, 48,64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288,304, 320, 336, 352, 368, 384, 409. 425, 441, 457, 473, 489, 505, 521,537, 553, 569, 585, 601, 617, 633, 649, 665, 681, 697, 713, 729, 745,761, 777, 793, 809, 825, 841, 857, 873 and 889, or a substantiallyidentical sequence thereof; and a light chain CDR3 selected from thegroup consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98, 11, 130, 146,162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370,386, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, 603,619, 635, 651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827,843, 859, 875 and 891, or a substantially identical sequence thereof. Ina preferred embodiment, the antibody or antibody fragment comprises thelight chain CDR1, CDR2 and CDR3 selected from the group consisting ofSEQ ID NO:439/441/443; 382/384/386; 791/793/795; and 807/809/811.

In a second aspect, the invention provides nucleic acid moleculesencoding the antibodies, or antigen-binding portions, of the invention.Recombinant expression vectors carrying the antibody-encoding nucleicacids of the invention, and host cells into which such vectors have beenintroduced, are also encompassed by the invention, as are methods ofmaking the antibodies of the invention by culturing the host cells ofthe invention.

In one embodiment, the antibody of the invention comprises a HCVRencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211,227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 396, 412, 428, 444,460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668,684, 700, 716, 732, 748, 764, 780, 796, 812, 892, 896, 900, 904, 908,912, 916, 920, 924, 934, 938, 942, and 946 or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a LCVRencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203,219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 404, 420, 436,452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660,676, 692, 708, 724, 740, 756, 772, 788, 804, 820, 894, 898, 902, 906,910, 914, 918, 922, 926, 936, 940, 944, and 948 or a substantiallysimilar sequence having at least 95% homology thereof.

In one embodiment, the antibody of the invention comprises a HCVRencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211,227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 396, 412, 428, 444,460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668,684, 700, 716, 732, 748, 764, 780, 796, 812, 892, 896, 900, 904, 908,912, 916, 920, 924, 934, 938, 942, and 946 or a substantially similarsequence having at least 95% homology thereof, and a LCVR encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 11,27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251,267, 283, 299, 315, 331, 347, 363, 379, 404, 420, 436, 452, 468, 484,500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708,724, 740, 756, 772, 788, 804, 820, 894, 898, 902, 906, 910, 914, 918,922, 926, 936, 940, 944, and 948 or a substantially similar sequencehaving at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR1 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85,101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309,325, 341, 357, 373, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542,558, 574, 590, 606, 622, 638, 654, 670, 686, 702, 718, 734, 750, 766,782, 798, 814, 830, 846, 862 and 878, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR2 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87,103, 119, 135, 151, 167, 183, 100, 215, 231, 247, 263, 279, 295, 311,327, 343, 359, 375, 400, 416, 432, 448, 464, 480, 496, 512, 528, 544,560, 576, 592, 608, 624, 640, 656, 672, 688, 704, 720, 736, 752, 768,784, 800, 816, 832, 848, 864 and 880, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR3 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 89,105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313,329, 345, 361, 377, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546,562, 578, 594, 610, 626, 642, 658, 674, 690, 706, 722, 738, 754, 770,786, 802, 818, 834, 850, 866 and 882, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a heavy chain CDR1 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85,101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309,325, 341, 357, 373, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542,558, 574, 590, 606, 622, 638, 654, 670, 686, 702, 718, 734, 750, 766,782, 798, 814, 830, 846, 862 and 878, or a substantially similarsequence having at least 95% homology thereof; a heavy chain CDR2encoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO:7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 100, 215,231, 247, 263, 279, 295, 311, 327, 343, 359, 375, 400, 416, 432, 448,464, 480, 496, 512, 528, 544, 560, 576, 592, 608, 624, 640, 656, 672,688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, 864 and 880, or asubstantially similar sequence having at least 95% homology thereof; anda heavy chain CDR3 encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361,377, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, 594,610, 626, 642, 658, 674, 690, 706, 722, 738, 754, 770, 786, 802, 818,834, 850, 866 and 882, or a substantially similar sequence having atleast 95% homology thereof. In a preferred embodiment, the antibody orantibody fragment comprises heavy chain CDR1, CDR2 and CDR3 encoded b anucleic acid sequence selected from the group consisting of SEQ IDNO:430/432/434; 373/375/377; 782/784/786; and 798/800/802.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR1 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93,109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317,333, 349, 365, 381, 406, 422, 438, 454, 470, 486, 502, 518, 534, 550,566, 582, 598, 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774,790, 806, 822, 838, 854, 870 and 886, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR2 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:15, 31, 47, 63, 79, 95,111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319,335, 351, 367, 383, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552,568, 584, 600, 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776,792, 808, 824, 840, 856, 872, and 888, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR3 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:17, 33, 49, 65, 81, 97,113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321,337, 353, 369, 385, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554,570, 586, 602, 618, 634, 650, 666, 682, 698, 714, 730, 746, 762, 778,794, 810, 826, 842, 858, 874 and 890, or a substantially similarsequence having at least 95% homology thereof.

In one embodiment, the invention features a human antibody or antibodyfragment comprising a light chain CDR1 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93,109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317,333, 349, 365, 381, 406, 422, 438, 454, 470, 486, 502, 518, 534, 550,566, 582, 598, 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774,790, 806, 822, 835, 851, 867 and 883, or a substantially similarsequence having at least 95% homology thereof; a light chain CDR2encoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207,223, 239, 255, 271, 287, 303, 319, 335, 351, 367, 383, 408, 424, 440,456, 472, 488, 504, 520, 536, 552, 568, 584, 600, 616, 632, 648, 664,680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, 856, 872, and888, or a substantially similar sequence having at least 95% homologythereof; and a light chain CDR3 encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 17, 33, 49, 65, 81, 97,113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321,337, 353, 369, 385, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554,570, 586, 602, 618, 634, 650, 666, 682, 698, 714, 730, 746, 762, 778,794, 810, 826, 842, 858, 874 and 890, or a substantially similarsequence having at least 95% homology thereof. In a preferredembodiment, the antibody or antibody fragment comprises the light chainCDR1, CDR2 and CDR3 encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:438/440/442; 381/383/385; 790/792/794; and806/808/810.

In a third aspect, the invention features an isolated antibody orantibody fragment that specifically binds hDII4, comprising a CDR 1, 2and 3 selected from the group consisting of (a) a heavy chain CDR1region comprising an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:928), wherein X¹ is Gly; X² is Phe orTyr; X³ is Thr; X⁴ is Phe; X⁵ is Ser, Thr or Asn; X⁶ is Ser, Asn or Tyr;X⁷ is Tyr or Phe; and X⁸ is Gly or Ala; (b) a heavy chain CDR2 regioncomprising an amino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸(SEQ ID NO:929), wherein X¹ is Ile or Leu; X² is Trp or Ser; X³ is Tyr,Ala or Gly; X⁴ is Asp, Ser or Tyr; X⁵ is Gly or Asp; X⁶ is Ser, Gly, Thror Val; X⁷ is Asn or Asp; and X⁸ is Lys or Arg; (c) a heavy chain CDR3region comprising an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶ (SEQ ID NO:930),wherein X¹ is Ala or Ser; X² is Arg or Lys; X³ is Asp or Tyr; X⁴ is Ser,Gly or His; X⁵ is Asp, Ala or Trp; X⁶ is Asn, or Phe; X⁷ is Tyr, Arg orLys; X⁸ is His or Ser; X⁹ is Gly or Trp; X¹⁰ is Tyr or Phe; X¹¹ is Gluor Asp; X¹² is Gly, His or Pro; X¹³ is Tyr, Trp or absent; X¹⁴ is Phe orabsent; X¹⁵ is Asp or absent; and X¹⁶ is Pro or absent.

In a preferred embodiment, the antibody or antibody fragment comprisesheavy chain CDR 1, 2 and 3 selected from the group consisting of (a) aheavy chain CDR1 region comprising an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:928), wherein X¹ is Gly; X² is Phe;X³ is Thr; X⁴ is Phe; X⁵ is Ser or Asn; X⁶ is Ser or Asn; X⁷ is Tyr orPhe; and X⁸ is Gly or Ala; (b) a heavy chain CDR2 region comprising anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ IDNO:929), wherein X¹ is Ile or Leu; X² is Trp or Ser; X³ is Tyr or Gly;X⁴ is Asp or Ser; X⁵ is Gly; X⁶ is Ser, Thr or Val; X⁷ is Asn or Asp;and X⁸ is Lys or Arg; (c) a heavy chain CDR3 region comprising an aminoacid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵X¹⁶ (SEQ ID NO:930),wherein X¹ is Ala or Ser; X² is Arg or Lys; X³ is Asp; X⁴ is Gly or His;X⁵ is Asp or Ala; X⁶ is Phe; X⁷ is Tyr or Arg; X⁸ is Ser; X⁹ is Gly; X¹⁰is Tyr; X¹¹ is Glu; X¹² is Gly or His; X¹³ is Tyr or Trp; X¹⁴ is Phe orabsent; X¹⁵ is Asp or absent; and X¹⁶ is Pro or absent.

In a further embodiment, the isolated antibody or antibody fragmentfurther comprises (d) a light chain CDR1 region comprising an amino acidsequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:931), wherein X¹is Gln; X² is Ser; X³ is Val; X⁴ is Arg, Ser or Thr; X⁵ is Ser or Gly;X⁶ is Ser or Tyr; and X⁷ is Tyr or absent; (e) a light chain CDR2 regioncomprising an amino acid sequence of the formula X¹-X²-X³ (SEQ IDNO:932), wherein X¹ is Gly or Asp; X² is Ala or Thr; and X³ is Ser; and(f) a light chain CDR3 region comprising an amino acid sequence of theformula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:933), wherein X¹ is Gln;X² is Gln or His; X³ is Tyr, Arg or Ser; X⁴ is Gly, Ser or Ala; X⁵ isSer, Asn or Phe; X⁶ is Trp or Ser; X⁷ is Pro; X⁸ is Trp, Pro or Arg; andX⁹ is Thr.

In a preferred embodiment, the isolated antibody or antibody fragmentfurther comprises (d) a light chain CDR1 region comprising an amino acidsequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:931), wherein X¹is Gln; X² is Ser; X³ is Val; X⁴ is Arg or Ser; X⁵ is Ser; X⁶ is Ser orTyr; and X⁷ is Tyr or absent; (e) a light chain CDR2 region comprisingan amino acid sequence of the formula X¹-X²-X³ (SEQ ID NO:932), whereinX¹ is Gly or Asp; X² is Ala or Thr; and X³ is Ser; and (f) a light chainCDR3 region comprising an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:933), wherein X¹ is Gln; X² is Glnor His; X³ is Tyr or Arg; X⁴ is Gly or Ser; X⁵ is Ser or Asn; X⁶ is Trpor Ser; X⁷ is Pro; X⁸ is Pro or Arg; and X⁹ is Thr.

In a fourth aspect, the invention features a fully human antibody orantibody fragment which binds hDII4 with an IC₅₀ of less than about 10nM, as measured in in vitro assay or ELISA-based DII4 blocking assay(described below). In a preferred embodiment, the antibody of theinvention exhibits an IC₅₀ of about 500 pM or less. In an even morepreferred embodiment, the antibody of the invention exhibits an IC₅₀ ofabout 100 pM or less.

In one embodiment, the invention provides a fully human monoclonalantibody which specifically binds and inhibits human DII4 and exhibitsan IC₅₀ of less than or equal to about 150 pM, 100 pM, 75 pM, or 50 pM,as measured by Notch-inducible luciferase bioassay with hDII4-Fc. Asshown in the experimental section below, the anti-hDII4 antibodies ofthe invention do not cross-react with closely related delta proteins,such as hDII1 and hDII3.

In one embodiment, the invention provides an isolated human antibody, oran antigen-binding portion thereof, that binds hDII4 with a K_(D) ofless than about 500 pM, preferably less than about 300 pM, even morepreferably less than about 100 pM, less than about 50 pM, less thanabout 10 pM, as determined by surface plasmon resonance (BIACORE™), forexample, using dimeric hDII4 (Table 2).

The invention encompasses anti-hDII4 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof a galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

The invention includes anti-hDII4 antibodies which bind specificepitopes of hDII4 and are capable of blocking the biological activity ofhDII4. The extracellular domain of DII4 is composed of an N-terminaldomain, a Delta/Serrate/Lag-2 (DSL) domain, and a tandem of eightepidermal growth factor (EGF)-like repeats. Generally, the EGF domainsare recognized as occurring at about amino acid residues 218-251 (domain1), 252-282 (domain 2), 284-322 (domain 3), 324-360 (domain 4), and362-400 (domain 5), with the DSL domain at about amino acid residues173-217 and the N-terminal domain at about amino acid residues 27-172 ofhDII4 (SEQ ID NO:2).

In one embodiment, a blocking antibody of the invention binds withinamino acids residues 27 to 524 of SEQ ID NO:2. In a more specificembodiment, a blocking antibody of the invention binds an epitope withinthe N-terminus-DSL domains 27-217 of SEQ ID NO:2; in an even morespecific embodiment, the blocking antibody binds an epitope within aboutamino acid residues 27-172 (N-terminal domain) or 173-217 (DSL domain).In another embodiment, a blocking antibody of the invention binds theEGF-2 epitope within about amino acids residues 252-282 of SEQ ID NO:2.

In a fifth aspect, the invention features a composition comprising arecombinant human anti-human DII4 antibody and an acceptable carrier.Further included in the invention are vectors and host cells comprisingvectors which contain nucleic acid molecules encoding the humananti-hDII4 antibody of the invention, as well as methods of producingthese novel antibodies, comprising growing a host cell comprisingnucleic acid encoding the anti-hDII4 antibody of the invention or anantibody fragment, under conditions permitting production of the proteinand recovering the protein so produced.

In a sixth aspect, the invention features methods for inhibiting hDII4activity using an antibody, or antigen-binding portion thereof, of theinvention. In one embodiment, the method comprises contacting hDII4 withthe instant antibody or antigen-binding portion thereof, such that hDII4is inhibited from binding to Notch receptor, for example Notch-1. Inanother embodiment, the method comprises administering an antibody orantibody fragment of the invention, to a human subject suffering from adisorder which is ameliorated by inhibition of DII4 activity. Thedisorder treated is a disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof DII4 activity, for example, pathological vascularization associatedwith tumor angiogenesis and cancer, immunodeficiency diseases,transplant rejection, or inflammation; and neurodegenerative conditions,e.g., associated with prion disease.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

DEFINITIONS

“Delta-like ligand 4”, “DII4”, “hDII4” are used interchangeably to referto the protein encoded by the nucleic acid sequence of SEQ ID NO:1 andthe protein having the amino acid sequence of SEQ ID NO:2.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementary determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “high affinity” antibody refers to those antibodies having abinding affinity to hDII4 of at least 10⁻⁸ M; preferably 10⁻⁹ M; evenmore preferably 10⁻¹⁰ M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate” or “Koff” is meant an antibody thatdissociates from hDII4 with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

A “neutralizing” or “blocking” antibody, is intended to refer to anantibody whose binding to DII4 results in inhibition of the biologicalactivity of DII4. This inhibition of the biological activity of DII4 canbe assessed by measuring one or more indicators of DII4 biologicalactivity. These indicators of DII4 biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see examples below). Preferably, the ability of an antibody toneutralize DII4 activity is assessed by inhibition of DII4 binding to aNotch receptor.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion” or “antibody fragment”), as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., hDII4). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 241:544-546), which consists of a VH domain;and (vi) an isolated CDR. Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. Other forms of single chain antibodies, such asdiabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl.Acad Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058).Antibody portions, such as Fab and F(ab′)₂ fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermlne immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlneimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germlne of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germlne immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germlne VH and VL sequences, may not naturally existwithin the human antibody germlne repertoire in vivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hDII4 is substantially free of antibodies that specifically bindantigens other than hDII4). An isolated antibody that specifically bindshDII4 may, however, have cross-reactivity to other antigens, such ashDII4 molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. In preferred embodiments, an antibody issaid to specifically bind an antigen when the equilibrium dissociationconstant is less than or equal to 10⁻⁸ M, more preferably when theequilibrium dissociation constant is less than or equal to 10⁻⁹ M, andmost preferably when the dissociation constant is less than or equal to10⁻¹⁰ M.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60, 70%, 80% or 90% W/W ofa protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide analog or variant” as used herein refers to apolypeptide that is comprised of a segment of at least 25 amino acidsthat has substantial identity to a portion of an amino acid sequence andthat has at least one of the following properties: (1) specific bindingto hDII4 under suitable binding conditions, or (2) ability to block DII4binding to a Notch receptor. Typically, polypeptide analogs or variantscomprise a conservative amino acid substitution (or insertion ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50, 60,70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often beas long as a full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton 1984 W. H. Freeman andCompany, New York; Introduction to Protein Structure (Branden & Tooze,eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature354:105, which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res.15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated herein by reference. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo et al.(1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

The term “percent sequence identity” in the context of nucleic acidsequences refers to the residues in two sequences which are the samewhen aligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides ormore, usually at least about 18 nucleotides, more usually at least about24 nucleotides, typically at least about 28 nucleotides, more typicallyat least about 32 nucleotides, and preferably at least about 36, 48 ormore nucleotides. There are a number of different algorithms known inthe art which can be used to measure nucleotide sequence identity. Forinstance, polynucleotide sequences can be compared using FASTA, Gap orBestfit, which are programs in Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis. FASTA, which includes, e.g., theprograms FASTA2 and FASTA3, provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences (Pearson (1990) Methods Enzymol. 183:63-98 and (2000) MethodsMol. Biol. 132:185-219, each herein incorporated by reference). Unlessotherwise specified, default parameters for a particular program oralgorithm are used. For instance, percent sequence identity betweennucleic acid sequences can be determined using FASTA with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) or using Gap with its default parameters as provided in GCGVersion 6.1, herein incorporated by reference.

A reference to a nucleic acid sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid molecule havinga particular sequence should be understood to encompass itscomplementary strand, with its complementary sequence. Generally, theart uses the terms “percent sequence identity”, “percent sequencesimilarity” and “percent sequence homology” interchangeably. In thisapplication, these terms shall have the same meaning with respect tonucleic acid sequences.

The term “substantial similarity”, or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, indicates that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 90%, preferably atleast about 95%, and more preferably at least about 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” or“substantially identical” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80% sequence identity, preferably at least90% or 95% sequence identity, even more preferably at least 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include 1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; 2)aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; and 6) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403 410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389 402, each of which is hereinincorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

Preparation of Human Antibodies

Methods for generating human antibodies include, for example,VELOCIMMUNE® (Regeneron Pharmaceuticals), XENOMOUSE™ technology(Abgenix), the “minilocus” approach, and phage display. The VELOCIMMUNE®technology (U.S. Pat. No. 6,596,541) encompasses a method of generatinga high specificity fully human antibody to a select antigen. Thistechnology involves generation of a transgenic mouse having a genomecomprising human heavy and light chain variable regions operably linkedto endogenous mouse constant region loci such that the mouse produces anantibody comprising a human variable region and a mouse constant regionin response to antigenic stimulation. The DNA encoding the variableregions of the heavy and light chains of the antibody are isolated andoperably linked to DNA encoding the human heavy and light chain constantregions. The DNA is then expressed in a cell capable of expressing thefully human antibody. In specific embodiment, the cell is a CHO cell.

The XENOMOUSE™ technology (Green et al. (1994) Nature Genetics 7:13-21)generates a mouse having both human variable and constant regions fromboth the heavy chain and kappa light chain loci. In an alternativeapproach, others have utilized a “minilocus” approach in which anexogenous Ig locus is mimicked through inclusion of individual genesfrom the Ig locus (see, for example, U.S. Pat. No. 5,545,807). The DNAencoding the variable regions can be isolated with or without beingoperably linked to the DNA encoding the human heavy and light chainconstant region.

Other methods of generating human antibodies, including isolation from ahuman donor, are known. See, for example, U.S. Pat. No. 6,787,637,herein specifically incorporated by reference in its entirety.

Antibodies may be therapeutically useful in blocking a ligand-receptorinteraction or inhibiting receptor component interaction, rather than bykilling cells through fixation of complement and participation in CDC.The constant region of an antibody is important in the ability of anantibody to fix complement and participate in CDC or direct cell killingthrough antibody-dependent cellular cytoxicity (ADCC). Thus, the isotypeof an antibody may be selected on the basis of the desirability for theantibody to fix complement.

Human immunoglobulins can exist in two forms that are associated withhinge heterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-heavy chain disulfidebonds and a molecule of about 75-80 kDa is formed composed of a singlelight and heavy chain. These forms have been difficult to separate, evenafter affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. In fact, asingle amino acid substitution in the hinge region of the human IgG4hinge can significantly reduce the appearance of the second form (Angalet al. 1993 Molecular Immunology 30:105) to levels typically observedusing a human IgG1 hinge. The instant invention encompasses antibodieshaving one or more mutations in the hinge, CH2 or CH3 region which maybe desirable, for example, in production to improve the yield, ormodulate effector functions.

Antibodies of the invention are preferably prepared with the use ofVELOCIMMUNE® technology. A transgenic mouse in which the endogenousimmunoglobulin heavy and light chain variable regions are replaced withthe corresponding human variable regions is challenged with the antigenof interest, and lymphatic cells (such as B-cells) recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloid-type cell line to prepare immortal hybridoma cell lines, andsuch hybridoma cell lines are screened and selected to identifyhybridoma cell lines that produce antibodies specific to the antigen ofinterest. DNA encoding the variable regions of the heavy chain and lightchain may be isolated and linked to desirable isotypic constant regionsof the heavy chain and light chain. Such an antibody protein may beproduced in a cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies may be isolated directly fromantigen-specific lymphocytes. In various embodiments, the transgenicmouse comprises 12 functional human variable heavy chain genes and 11functional human variable kappa light chain genes; 25 to 30 humanvariable heavy chain genes and from 18 to 20 human variable kappa lightchain genes; 43 to 48 human variable heavy chain genes and 20 to 22human variable kappa light chain genes; or about 80 human variable heavychain genes and about 40 human variable kappa light chain genes.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻⁹ through about10⁻¹¹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention, for example wild-type or modified IgG1 or IgG4 (forexample, SEQ ID NO:950, 951, or 952). While the constant region selectedmay vary according to specific use, high affinity antigen-binding andtarget specificity characteristics reside in the variable region.

Cancer, infectious diseases, autoimmunity, immunodeficiency,transplants, inflammation, injury and degenerative conditions can betreated by modulation of the immune system. In cases of disease due toinappropriate function or hyperactivity of the immune system, such asautoimmunity or inflammation, can be ameliorated through inhibition ofimmune cell function or reduction of immune cell numbers. This can beaccomplished by blockade of positive signals or stimulation of negativesignals on immune cell populations critical to the disease process, suchas T, B or NK cells, neutrophils, macrophages, antigen presenting cells,mast cells or other cell types. Overactivity can also be inhibitedthrough elimination of various immune cell populations by stimulation ofapoptosis, targeting of specific surface receptors with depletingantibodies or antibody-drug conjugates, or the blockade or alteration ofthe differentiation of immune cell lineages or specific cell types.Inefficient or reduced immune function can cause or exacerbate disorderssuch as cancer, infectious disease, and other immunodeficiencies.Hypoactivity of the immune system can be improved through activation ofimmune cells by stimulation of positive signals by crosslinking oragonistic antibodies or blockade of negative signals. Immune cellpopulations can be increased by stimulation of development of some orall immune cell lineages, prevention of apoptosis, or elimination ofinhibitory signals. In a specific application, the antibodies of theinvention are useful for treatment, inhibition or amelioration of acondition or disease such as, for example, cancer, immunodeficiency,transplant rejection, or inflammation.

Epitope Mapping and Related Technologies

To screen for antibodies which bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described in Harlow and Lane (1990)supra can be performed. Other methods include alanine scanning mutants,peptide blots (Reineke (2004) Methods Mol Biol 248:443-63) (hereinspecifically incorporated by reference in its entirety), or peptidecleavage analysis. In addition, methods such as epitope excision,epitope extraction and chemical modification of antigens can be employed(Tomer (2000) Protein Science 9: 487-496) (herein specificallyincorporated by reference in its entirety).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (U.S.patent Publication No. 2004/0101920, herein specifically incorporated byreference in its entirety). Each category may reflect a unique epitopeeither distinctly different from or partially overlapping with epitoperepresented by another category. This technology allows rapid filteringof genetically identical antibodies, such that characterization can befocused on genetically distinct antibodies. When applied to hybridomascreening, MAP may facilitate identification of rare hybridoma clonesthat produce mAbs having the desired characteristics. MAP may be used tosort the hDII4 antibodies of the invention into groups of antibodiesbinding different epitopes.

Agents useful for altering the structure of the immobilized antigen areenzymes, such as, for example proteolytic enzymes, for example, trypsin,endoproteinase Glu-C, endoproteinase Asp-N, chymotrypsin, etc. Agentsuseful for altering the structure of the immobilized antigen may also bechemical agents, such as, succinimidyl esters and their derivatives,primary amine-containing compounds, hydrazines and carbohydrazines, freeamino acids, etc.

The antigen protein may be immobilized on either biosensor chip surfacesor polystyrene beads. The latter can be processed with, for example, anassay such as multiplex LUMINEX™ detection assay (Luminex Corp., Austin,Tex.). Because of the capacity of LUMINEX™ to handle multiplex analysiswith up to 100 different types of beads, LUMINEX™ provides almostunlimited antigen surfaces with various modifications, resulting inimproved resolution in antibody epitope profiling over a biosensorassay.

Therapeutic Administration and Formulations

The administration of therapeutic entities in accordance with theinvention will be administered with suitable carriers, excipients, andother agents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences (15th ed, Mack PublishingCompany, Easton, Pa.). These formulations include, for example, powders,pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic oranionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. Any of theforegoing mixtures may be appropriate in treatments and therapies inaccordance with the present invention, provided that the activeingredient in the formulation is not inactivated by the formulation andthe formulation is physiologically compatible and tolerable with theroute of administration. See also Powell et al. “Compendium ofexcipients for parenteral formulations” PDA (1998) J Pharm Sci Technol.52:238-311 and the citations therein for additional information relatedto excipients and carriers well known to pharmaceutical chemists.

EXAMPLES Example 1 Generation of Human Antibodies to Human DII4

Mice may be immunized by any method known in the art (see, for example,Harlow and Lane supra). In one embodiment, hDII4 antigen is administereddirectly to VELOCIMMUNE® mice comprising DNA loci encoding human Igheavy chain variable regions and kappa light chain variable regions,with an adjuvant to stimulate the immune response. Such an adjuvantincludes complete and incomplete Freund's adjuvant, MPL+TDM adjuvantsystem (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan 2000 VaccineAdjuvant, by Human Press, Totawa, N.J.). The antibody immune response ismonitored by standard antigen specific immunoassay. When a desiredimmune response is achieved, antibody-expressing B cells were harvestedand fused with mouse myeloma cells to preserve their viability, forminghybridoma cell lines. Such hybridoma cell lines are screened andselected to identify cell lines that produce antigen-specific antibodiesusing assays as described below.

Alternatively, antigen-specific hybridoma cells may be isolated by flowcytometry. Briefly, after fusion to myeloma cells, pooled hybridomacells were grown for 10 days in HAT medium. The cells were thenharvested and stained with biotin-labeled DII4 at 2 mg/ml for one hour,followed by addition of phycoerythrin-streptavidin. Thefluorescence-labeled cells were sorted by flow cytometry (single cellper well into 96 well plates containing hybridoma growth medium),cultured for 8-10 days, and conditioned media screened for the presenceof functionally desirable monoclonal antibodies, as described below.

Anti-hDII4 antibodies generated via direct isolation of splenocytes.Antigen-specific antibodies may also be isolated directly fromantigen-immunized B cells without fusion to myeloma cells, as describedin U.S. Patent Publication 2007/0280945A1, herein specificallyincorporated by reference in its entirety. Stable recombinantantibody-expressing CHO cell lines were established from the isolatedproper recombinants.

Example 2 Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D) values) for antigen binding tothe selected antibodies described above were determined by surfacekinetics on a real-time biosensor surface plasmon resonance assay(BIACORE™ 2000). The antibody was captured on a goat anti-mouse IgGpolyclonal antibody surface created through direct chemical coupling toa BIACORE™ chip to form a captured antibody surface. Varyingconcentrations of monomeric hDII4 or dimeric hDII4-hFc were injectedover the captured antibody surfaces, and antigen-antibody binding anddissociation monitored in real time. Kinetic analysis was performed tocalculate K_(D), dissociation rate constants, and half-life ofantigen/antibody complex dissociation (Table 1). A similar method wasapplied to measure single B cell-derived monoclonal antibodies modifiedto contain a human IgG constant domain. Antibodies were presented bygoat anti-hFc polyclonal antibody reagent (Jackson Immuno Research Lab)immobilized on BIACORE™ chip, and exposed to either dimeric DII4-mFc ormonomeric DII4 protein (Table 2).

Antibody-antigen binding affinity may also be assessed using an ELISAbased solution competition assay. Briefly, on a 96-well microtiterplate, antibodies (purified proteins or in conditioned medium) werepremixed with serial dilutions of antigen protein (monomeric or dimeric)ranging from 0 to 10 μg/ml with a constant concentration of antibody.After a 2 hr incubation of antigen with antibody, the solutions weretransferred to a microtiter plate precoated with antigen for measurementof free antibody (MAXISORB™, VWR, West Chester, Pa.). The plate wascoated with 1 μg/ml hDII4-hFc protein in PBS solution overnight at 4° C.and nonspecific binding sites blocked with BSA for 2 hrs. After a 1 hrincubation following transfer, the plate was washed and the plate-boundantibodies were detected with an HRP-conjugated goat anti-mouse IgGpolyclonal antibody reagent (Jackson Immuno Laboratory) and developedusing colorimetric substrates (OPTEIA™; BD Biosciences Pharmingen, SanDiego, Calif.). The enzymatic reaction was stopped with 1 M phosphoricacid, optical absorptions at 450 nm were recorded and the data wereanalyzed using a sigmoidal dose-response model and an IC₅₀ values werereported (Table 1).

TABLE 1 Antibody K_(D) DII4 (nM) K_(D) DII4-Fc (nM) IC₅₀ DII4-Fc (nM)13B6 2.79 0.188 0.06 15E10 0.55 0.023 0.58 22G12 1.29 0.076 0.03 24C80.52 0.047 0.01 VAV 2H4-19 1.51 0.611 0.10 VAV 4H10-9 13.70 0.662 0.30VAV 7B9-9 0.88 0.021 0.27 VAW 10E4-9 89.00 0.468 0.06 VAW 10G11-2 31.301.430 1.66 VAW 1C6-1 45.80 0.092 0.25 VAW 1G2-4 83.80 0.035 0.40 VAW1H2-2 67.00 0.148 0.30 VAW 2H3-2 0.30 0.150 0.26 VAW 3A7-2 1.64 0.1620.02 VAW 3A9-5 NA 2.510 16.00 VAW 3F12-8 8.12 0.648 0.07 VAW 6B8-12 0.890.060 0.43 VAW 6C6-2 91.70 0.092 0.50 VAW 6G12-10 3.74 0.527 0.19 VAW7C10-11 17.10 0.853 0.28 VAW 8A10-14 1.41 0.648 0.08 VAW 8G1-12 6.098.300 8.60 VAW 9B11-2 62.20 0.048 0.00 VAW 9F12-6 16.00 1.350 0.02 VAW9G10-1 56.10 0.555 0.10

TABLE 2 Antibody K_(D) DII4 (nM) K_(D) DII4-Fc (nM) 314266-06F12-B7 2.170.075 318518-01A04-D5 0.237 0.244 318518-01A10-D8 0.399 0.018318518-01B09-C3 0.833 0.180 318518-01B11-D4 0.382 0.088 318518-01E07-H20.165 0.238 318518-01G04-F3 0.501 0.107 318518-01G05-B5 1.06 0.196318518-02A07-B3 0.208 0.148 318518-02B06-E2 2.15 0.193 318518-02B08-F7N/A N/A 318518-02C04-D1 0.478 0.331 318518-02F05-D10 1.28 0.035318518-02G03-F2 1.31 0.042 318518-02G04-B11 0.813 0.048 318518-02G08-F11N/A N/A 318518-03A03-B2 0.136 0.124 318518-03C10-F2 1.18 0.131318518-03D04-B5 0.904 0.136 318518-03D07-G11 3.74 0.163 318518-03F04-A60.501 0.088 318518-03F06-A3 0.556 0.037 318518-03H03-F3 8.89 0.084318518-14A06-E7 4.54 0.282 318518-14A07-C4 0.235 0.035 318518-14D08-G10.541 0.046 318518-14H08-A2 6.67 0.128 318518-1H08-E9 0.225 0.050

Example 3 Inhibition of DII4 and Notch Interaction

The ability of the antibodies to block DII4 binding to Notch wasevaluated with an ELISA-based immunoassay. Briefly, Notch-hFcrecombinant protein was coated on a 96-well plate in PBS bufferovernight at 4° C. at 1 mg/ml, and the nonspecific binding sites wereblocked with BSA. This plate was used to measure free biotin-DII4-hFcfrom antibody titration sample solutions. To make the antibody titrationsamples, a constant amount of biotin-DII4-hFc at 25 pM was pre-mixedwith varied amounts of antibody, either in crude hybridoma conditionmedium or as purified antibody protein, ranging from 0 to 50 nM inserial dilutions, followed by 2 hr incubation at room temperature toallow antibody-antigen binding to reach equilibrium. The equilibratedsample solutions were then transferred to the Notch-hFc coated platesfor the measurement of free biotin-DII4-hFc. After 1 hour binding, theplate was washed and bound biotin-DII4-hFc was detected using HRPconjugated streptavidin (Poly HRP streptavidin, Pierce Endogen), anddeveloped using TMB substrate (BD Pharmigen). Data was analyzed usingGraphPad Prism software and IC₅₀ values were determined as the amount ofantibody required to achieve 50% reduction of biotin-DII4-hFc bound tothe plate-coated Notch-Fc (Table 3) (*conditioned media).

TABLE 3 Antibody IC₅₀ (nM) VAV 2H4-19 0.01 VAW 3A7-2 0.017 VAW 9G10-10.019 VAW 10E4-9 0.032 VAW 8A10-11 0.04 VAW 9F12-6 0.059 VAW 3F12-80.066 VAW 1C6-1 0.086 VAW 1G2-4 0.11 VAW 6C6-2 0.119 VAV 7B9-4 0.123 VAW1H2-2 0.154 VAW 2H3-2 0.168 VAW 6B8-12 0.255 VAW 6G12-10 0.257 VAW7C10-11 0.273 VAV 4H10-9 0.599 VAW 10G11-2 0.931 VAW 3A9-5 3.8 VAW8G1-12 10.7 VAW 9B11-2 0.069 15E10* 0.04 22G12 0.10 13B6 0.11 24C8 0.031314266-06F12-B7 0.07 318518-01A04-D5 0.11 318518-01A10-D8 0.05318518-01B09-C3 0.03 318518-01B11-D4 0.03 318518-01E07-H2 1.17318518-01G04-F3 0.02 318518-01G05-B5 1.08 318518-02A07-B3 0.03318518-02B06-E2 0.09 318518-02B08-F7 N/A 318518-02C04-D1 0.60318518-02F05-D10 0.16 318518-02G03-F2 0.07 318518-02G04-B11 1.09318518-02G08-F11 N/A 318518-03A03-B2 0.57 318518-03C10-F2 0.12318518-03D04-B5 0.04 318518-03D07-G11 0.45 318518-03F04-A6 0.01318518-03F06-A3 0.02 318518-03H03-F3 0.17 318518-14A06-E7 0.04318518-14A07-C4 0.02 318518-14D08-G1 0.14 318518-14H08-A2 0.25318518-1H08-E9 0.03

The ability of selected purified anti-hDII4 antibodies to block DII4binding to Notch was also evaluated with the ELISA-based immunoassaydescribed above, modified by replacing 25 pM of biotin-DII4-hFc with 30pM of biotin-DII4-hFc, and reducing antibody-antigen incubation durationfrom 2 hrs to 1 hr. For convenience, antibody 318518-01A10-D8 wasrenamed “REGN281” (HCVR/LCVR SEQ ID NOs:429/437 and hIgG1 SEQ IDNO:950). Derived antibodies tested included REGN421 (HCVR/LCVR SEQ IDNO:901/903, hIgG1 SEQ ID NO:950); and REGN422 (HCVR/LCVR SEQ IDNO:901/903, with modified hIgG4 SEQ ID NO:952). Results are shown inTable 4.

TABLE 4 Antibody IC₅₀ (nM) REGN281 0.042 REGN421 0.045 REGN422 0.039

Ability of antibody to neutralize DII4 mediated cellular function wasalso tested in vitro using DII4 expressing human umbilical veinendothelial cells (HUVEC). Inhibition of Notch mediated hHes1 and EphB2gene expression in HUVEC with the derived antibodies was monitored asfollows: low passage HUVEC were cultured in MCDB-131 media (VecTechnologies). One day prior to analysis, HUVEC cells were seeded at adensity of 2×10⁵ cells/well in 24-well plates in 1 ml total mediavolume. The test antibody or other inhibitor was added directly to theindividual sample wells in triplicate followed by 5-hour culture at 37°C. At the end of the culture period, media was removed and total RNA wasisolated using QIAZOL™ and the RNEASY™ lipid tissue kit (Qiagen). mRNAlevel quantification was performed by the use of PCR and the fluorogenic5′ nuclease assay (TAQMAN® assay, Appied Biosystems). For each sample,cDNA was synthesized from 1-2 mg of total RNA. cDNA generated from anequivalent amount of starting RNA (typically 25 ng) was loaded intriplicate on ABI PRISM™ optical reaction plates. For each RNA sample a“no RT” control was also run in which no reverse transcriptase was addedto allow for subtraction of any potential genomic DNA contributions tothe signal. 2× Mastermix (TAQMAN® 2×PCR Mastermix; ABI) was added toeach reaction to a final concentration of 1×. Additionally, TAQMAN®probe and primers for the gene of interest were added to each reaction.Each primer was used at a final concentration of 900 nM and the probewas added at a final concentration of 200 nM. Human genomic DNA was usedas a standard. The assays were performed under standard TAQMAN®conditions on a ABI 7900HT instrument. Levels of Hes1 and EphrinB2 weremeasured and normalized to an endogenous control gene (cyclophilin)(Table 5). Probes and primers: human Hes1 probe (SEQ ID NO:387); Oligos:hHes1-869F (SEQ ID NO:388); hHes1-940R (SEQ ID NO:389), human ephrinB2probe: hEphB2-773T (SEQ ID NO:390); Oligos: hEphB2-752F (SEQ ID NO:391)hEphB2-812R (SEQ ID NO:392); human cyclophilin: probe: hCyclophilin-343T(SEQ ID NO:393); Oligos: hCyclophilin-323F (SEQ ID NO:394);hCyclophilin-389R (SEQ ID NO:395).

TABLE 5 EphrinB2 Expression Hes1 Expression Antibody IC₅₀ (nM) IC₅₀ (nM)22G12 0.379 0.381 15E10 2.56 4.49 VAW 3A7-2 0.409 0.533 314266-06F12-B70.239 0.405 318518-01A10-D8 0.305 0.329 318518-01G04-F3 0.088 0.172318518-01H08-E9 0.413 0.548 318518-02A07-B3 0.398 0.128 318518-03F04-A60.158 0.115 318518-03F06-A3 0.304 0.692 318518-014A07-C4 0.175 0.312318518-014D08-G1 0.510 0.568 hDII4-hFc 0.843 0.974

HUVEC Proliferation Assay. The ability of antibody to block DII4mediated growth inhibition of Human umbilical vein endothelial cells(HUVEC) cells was tested in an in vitro cell proliferation assay. Lowpassage HUVEC cells were obtained and cultured in MCDB-131 media (VecTechnologies). One day prior to analysis 12-well tissue culture plateswere coated with hDII4-hFc in PBS at 4° C. overnight (0.2 μg/ml; 0.5 mlPBS/well). Plates were washed 1× with PBS and HUVEC cells were seeded ata density of 4×10³ cells/well in 1.0 ml total media volume. Immediatelyfollowing addition of cells anti-hDII4 antibodies were added in 0.5 mltotal volume over a range of concentrations to generate an inhibitioncurve. Cells were grown for 96-hours at 37° C. Cell number wasquantitated using the CCK-8 reagent (Dojindo). All assays were run intriplicate. (Table 6, NB, not blocking).

TABLE 6 Antibody IC₅₀ (nM) 15E10 0.284 VAW9B11-2 1.868 VAW8D8-12 NB 13B65.01 VAW2H4-19 NB VAW3A7-2 0.198 VAW8A10-14 0.214 22G12 0.888318518-06F12-B7 2.067 318518-01A10-D8 0.096 318518-01G04-F3 0.106318518-01H08-E9 0.188 318518-02A07-B3 0.200 318518-03F04-A6 0.184318518-03F06-A3 0.188 318518-014A07-C4 0.159 318518-014D08-G1 0.165

Notch-Inducible Luciferase Assay. A bioassay was developed to determinethe ability of selected purified antibodies to neutralize DII4-mediatedcellular function in vitro using an engineered HEK293 cell line (ATCC)that constitutively expresses human Notch 1 and contains aNotch-responsive promoter driving luciferase. Inhibition ofNotch-inducible luciferase activity was determined as follows: 1 dayprior to assay, each well of an opaque 96 well tissue culture plate wascoated with 100 μl of either 1 nM or 1.5 nM hDII4-hFc in PBS overnightat 4° C. Cells were seeded onto the coated plates at 2×10⁴ cells/well inmedia. Purified antibody protein, in serial dilutions starting from 2 nMin cell media, was incubated with the cells at 37° C. for 24 hrs.Luciferase activity was determined by adding an equal well volume ofSTEADY-GLO® Substrate (Promega) (Table 7).

TABLE 7 IC₅₀ (pM) Antibody 1 nM hDII4-hFc 1.5 nM hDII4-hFc REGN281 50.5 78.7 REGN421 54.4  87.3 REGN422 88.2 131.1

Example 4 Inhibition of Notch1 Cleavage

The ability of selected anti-hDII4 antibodies to inhibit Notch1 cleavagewas tested by examination of total cleaved Notch1 protein bySDS-Page/Western blotting. Low passage HUVEC cells were cultured asdescribed above. One day prior to analysis 6-well plates were coatedwith hDII4-hFc in PBS at 4° C. overnight (0.2 μg/ml; 1.0 ml PBS/well).Plates were washed 1× with PBS and HUVEC cells were seeded at 7.5×10⁵cells/well in 2.0 ml total media volume. Immediately following cellseeding, anti-hDII4 antibody was added to each well at 10 nM finalconcentration. Cells were grown for 24 hours at 37° C. following whichwhole cell extracts were prepared and analyzed by SDS-PAGE. Levels ofcleaved Notch1 were determined using an anti-cleaved Notch1 (Val1744)antibody (Cell Signaling) and standard western blotting techniques. Theanti-hDII4 antibodies were able to entirely block the Notch1 cleavageinduced by plate coated hDII4-hFc (data not shown).

Example 5 ADCC and CDC Assays

Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by two testantibodies (REGN421, REGN422) was assessed using a panel of eight targetcell lines with varying hDII4 expression levels. The eight target celllines were (1) HUVECs; (2) HUVECs stimulated with 10 nM VEGF for 24hours; (3) Colo205; (4) engineered C6 rat glioma cells expressing eGFP;(5) engineered C6 rat glioma cells expressing hDII4; (6) engineeredHT1080 cells expressing eGFP; (7) engineered HT1080 cells expressinghDII4; and (8) HT29. Human DII4 or eGFP was integrated into the C6 cellor HT1080 genome through retroviral transfection. Briefly, cells fromeach target cell line (10,000 cells/well in 50 μl) were first mixed withan equal volume of serially diluted REGN421 or REGN422, resulting in afinal antibody concentration ranging from 0.169 pM to 10 nM, andincubated for 10 min at room temperature in a 96-well plate format(control=wells without antibody). Separately, human peripheral bloodmononuclear cells (PBMCs, effector cells) were prepared following aconventional Ficoll-Hypaque gradient centrifugation enrichmentprocedure. Approximately 300,000 PBMCs were added to each mixture ofantibody and target cells to give a final ratio of effector to targetcells of approximately 30:1. The 96-well plates were then incubated for4 h at 37° C., 5% CO₂ followed by centrifugation at 250×g. Supernatantswere harvested and assayed for lactate dehydrogenase (LDH) activityusing the CYTOTOX 96® Non-Radioactive Cytotoxicity Assay system(Promega). Results are shown in Table 8. REGN421-induced dose dependentcell lysis was only observed in C6 cells expressing hDII4 (col. 5),which exhibited the highest hDII4 expression among all cell lines (asdetermined by immunoprecipitation/Western blot and flow cytometry). Themaximum cell cytotoxicity in the C6-hDII4 cell line ranged from 20% to60%. No REGN421-induced cell lysis was observed in the remaining seventarget cell lines. REGN422 did not induce cell lysis in any of thetarget cell lines.

TABLE 8 % Maximum Cytotoxicity Ab 1 2 3 4 5 6 7 8 REGN421 0 0 0 0 20-600 0 0 REGN422 0 0 0 0 0 0 0 0

Complement-dependent cytotoxicity (CDC) induced by REGN421 was assessedusing the same panel of cells lines described above. Briefly, cells fromeach of the target cell lines (50,000 cells/well in 50 μl) were firstmixed with an equal volume of serially diluted REGN421, resulting in afinal antibody concentration ranging from 0.169 pM to 10 nM, andincubated for 10 min at room temperature in a 96-well plate format.Normal human serum, with complement components (Quidel Corp., San Diego,Calif.) was added to each well to give a final serum concentration of5%. The plates were then incubated at 37° C., 5% CO₂ for 2 hoursfollowed by addition of CELLTITER-BLUE® reagent (Promega)(controls=wells without antibody and wells with antibody but no serum).The plates were incubated overnight and cell survival (CDC levels)assayed. As a positive control, Daudi cells were treated with rituximab.REGN421 exhibited no CDC toward any of the target cell lines tested(data not shown).

Example 6 Epitope Mapping and Specificity

In order to determine epitope binding specificity, a series of sevenchimeric DII4 proteins were generated in which specific human DII4domains were substituted into a mouse DII4 protein as follows: #1contained the human N-terminal and DSL domains (S27-Q218); #2 containedhuman N-terminal, DSL and EGF-1 domains (S27-N252); #3 contained humanN-terminal, DSL, EGF-1 and EGF-2 domains (S27-Q283); #4 contained humanN-terminal, DSL, EGF-1, EGF-2, EGF-3, EGF-4 and EGF-5; #5 containedhuman N-terminal domain (S27-R172); #6 contained human DSL domain(V173-Q218); and #7 contained human EGF-2 domain (E252-D282). Thechimeric proteins were fused to a mouse IgG2a-Fc fragment and expressedin CHO-K_(i) cell. The conditioned media were harvested and proteinexpression confirmed by western blot.

Binding specificity of test antibodies to hDII4, mDII4, and chimericproteins #1, #2, #3, and #4 were tested as follows: purified antibodies22G12, VAW3A7-2, and 15E10 were amine coupled between 5000-6000 RU onCM5 chip. Conditioned media from CHO K1 cells containing the chimericDII4 proteins, hDII4-mFc, and mDII4-mFc were injected sequentiallyfollowed by surface regeneration over antibody-coupled surfaces. A blankamine coupled flowcell surface was used as a control for nonspecificbinding of the conditioned media. Results are summarized in Table 9.22G12 bound an epitope between S27-Q218 of hDII4; VAW3A7-2 bound anepitope between Q283-E400 hDII4; and 15E10 bound an epitope betweenE252-D282 of hDII4.

TABLE 9 Chimeric Proteins Antibody hDII4-mFc mDII4-mFc #1 #2 #3 #422G12 + − + + + + VAW3A7-2 + − − − − + 15E10 + − − − + +

Binding specificity of purified test mAb to hDII4, mDII4 and thechimeric proteins (described above) was determined(REGN279=314266-6F12-B7; REGN287=318518-1G04-F3; REGN289=318518-1H08-E9;REGN290=318518-2A07-B3; REGN306=318518-3F06-A3). Briefly, each DII4protein was captured (70-130 RU) on goat anti-mouse IgG antibodysurfaces, followed by injection of test mAb at a concentration of 100μg/ml. An antibody that bound mDII4-mFc was used as a positive control(positive control=6C10). The results (Table 10) show that REGN279 boundan epitope between S27-Q218 of hDII4; REGN287 bound between Q283-E400 ofhDII4; REGN289, REGN290 and REGN306 bound between S27-E400 of hDII4.

TABLE 10 Chimeric human-mouse DII4 Fusion Proteins Antibody hDII4-mFcmDII4-mFc #1 #2 #3 #4 #5 #6 #7 REGN279 + − + + + + + − − REGN287 + − − −− + − − − REGN289 + − + + + + − + − REGN290 + − + + + + − + − REGN306 +− + + + + − + − Control + + + + + + + + +

Further epitope binding specificity determinations were conducted asdescribed above with the following purified test antibodies:REGN281=318518-1A10-D8; REGN305=318518-3F04-A6; REGN309=318518-14A07-C4;REGN310=318518-14D08-G1; REGN421, and REGN422. Briefly, each of the DII4proteins was captured (240-470 RU) on goat anti-mouse IgG antibodysurfaces, followed by injection of test antibody at a concentration of100 μg/ml (Table 11).

TABLE 11 Chimeric human-mouse DII4 Fusion Proteins Antibody hDII4-mFcmDII4-mFc #1 #2 #3 #4 #5 #6 #7 REGN281 + − + + + + + + − REGN305 + − − −− + − − − REGN309 + − + + + − + + − REGN310 + − − − + + − + + REGN421 +− + + + + + + − REGN422 + − + + + + + + −

Western blot analysis. Binding specificity of selected antibodies tochimeric, mouse and human DII4 was determined by Western blot. Briefly,hDII4-mFc (200 ng per lane), mDII4-mFc (200 ng per lane), and chimericproteins #1-#7 (approximate 150 ng per lane) were subjected toelectrophoresis on duplicate SDS-PAGE gels using non-reducing samplebuffer. Each gel was then transferred to a PVDF membrane. Blots werefirst exposed to REGN421 at 0.2 μg/ml and then to HRP-conjugatedanti-hIgG antibody (Pierce). Control blots were exposed toHRP-conjugated anti-mFc antibody (Pierce). Results: REGN421 recognizedhDII4-mFc and chimeric proteins containing the human N-terminal domain(#5), a human DSL domain (#6), or both (#1, #2, #3, and #4). REGN421 didnot recognize a chimeric protein containing a human EGF-2 domain (#7).

Protease digestion analysis. Binding between REGN281 and hDII4 wasfurther assessed by protective protease digestion and liquidchromatography/mass spectrometry (LC/MS) using an HPLC1100 (Agilent) andLCQ Classical Ion Trap Mass Spectrometer (Thermo). Briefly, a mixture ofhDII4 and REGN281, in a molar ratio of 1:5, or hDII4 alone, wasincubated with protease overnight at either 25° C. (for GluC protease)or 37° C. (for trypsin). Each of the resulting proteolytic digestmixtures was then subjected to LC/MS. Unique peptide peaks present inproteolytic digests performed in the absence of REGN281, which eitherdiminished or disappeared in proteolytic digests performed in thepresence of REGN281, indicate potential REGN281 binding sites on hDII4that were protected from protease digestion by the binding of REGN281 tohDII4. These unique peptide peaks were analyzed by mass spectrometry.The observed mass, predicted mass, and the N-terminal sequences of thepeptides are shown in Table 12.

TABLE 12 Observed Predicted hDII4 Peptide Peak Mass Mass (SEQ ID NO:2)Protease Domain G1 521 521.5 Phe37-Glu40 GluC N-terminal G2 1362.81363.4 Ala121-Glu132 GluC N-terminal T1 758 760.8 Pro49-Arg55 TrypsinN-terminal T2 587.1 587.2 Tyr169-Arg172 Trypsin N-terminal T3 1607.41607.6 Val173-Arg186 Trypsin DSL T4 1399 1400.7 Gly42-Arg55 TrypsinN-terminal T5 569.2 569.3 Thr56-Arg59 Trypsin N-terminal T7 2615 2613.4Ile143-Arg166 Trypsin N-terminal T8 1807.2 1806.9 Ser27-Arg41 TrypsinN-terminal

Example 7 Binding Affinity of Purified Antibodies to Human and MonkeyDII4

The binding affinities of selected purified antibodies to hDII4, M.facscicularis DII4 (mfDII4, SEQ 1N NO:956), and M. mulatta DII4 (mmDII4,SEQ ID NO:957) monomers were determined using a BIACORE™ 2000 & 3000.Goat anti-hFc polyclonal antibody reagent immobilized on a BIACORE™ chipwas used to present REGN281, REGN421, and REGN422. Varyingconcentrations of each proteins, hDII4 (from 12.5 nM to 100 nM), mfDII4(from 3.13. nM to 100 nM), or mmDII4 (from 12.5 nM to 100 nM) were usedas analyte, and injected over the antibody surfaces. Antigen-antibodybinding and dissociation of bound complex were monitored in real time(Table 13).

TABLE 13 ka (M-1s-1) kd (s-1) K_(D) (nM) hDII4 mfDII4 mmDII4 hDII4mfDII4 mmDII4 hDII4 mfDII4 mmDII4 REGN281 1.56 × 10⁵ 6.64 × 10⁴ 9.27 ×10⁴ 2.30 × 10⁻⁵ 2.04 × 10⁻⁵ 3.05 × 10⁻⁵ 0.148 0.307 0.329 REGN421 1.63 ×10⁵ 7.28 × 10⁴ 9.70 × 10⁴ 2.17 × 10⁻⁵ 2.02 × 10⁻⁵ 3.23 × 10⁻⁵ 0.1330.278 0.333 REGN422 1.64 × 10⁵ 8.01 × 10⁴ 9.27 × 10⁴ 2.36 × 10⁻⁵ 2.88 ×10⁻⁵ 3.41 × 10⁻⁵ 0.144 0.360 0.375

The binding affinities of anti-hDII4 antibodies toward hDII4 and mmDII4dimers were also determined using a BIACORE™ 2000 and the methoddescribe above, except that hDII4 was replaced with hDII4-mFc (from 3.13nM to 100 nM), or mmDII4 with mmDII4-mFc (from 0.78 nM to 25 nM) asanalyte (Table 14).

TABLE 14 ka (M-1s-1) kd (s-1) K_(D) (nM) hDII4-mFc mmDII4-mFc hDII4-mFcmmDII4-mFc hDII4-mFc mmDII4-mFc REGN281 3.02 × 10⁵ 3.16 × 10⁵ 4.96 ×10⁻⁶ 4.64 × 10⁻⁶ 0.0163 0.0147 REGN421 3.43 × 10⁵ 3.35 × 10⁵ 4.70 × 10⁻⁶3.80 × 10⁻⁶ 0.0137 0.013  REGN422 3.46 × 10⁵ 4.23 × 10⁵ 4.60 × 10⁻⁶ 4.15× 10⁻⁶ 0.0133 0.0098

Example 8 Cross-Reactivity of Antibodies with hDII1, hDII3, mDII4, ormfDII4

Cross-reactivity of the antibodies to human delta-like ligand1 (SEQ IDNO:953) and human delta-like ligand 3 (SEQ ID NO:954) proteins wasdetermined. REGN281, REGN421 and REGN422 were presented by a goatanti-human kappa (hK) polyclonal antibody reagent (Southern Biotech)immobilized on a BIACORE™ chip, and either hDII4-hFc or hDII1-hFcprotein at 100 μg/ml were used as analyte injected over the antibodysurfaces. All three anti-hDII4 antibodies only bound to hDII4-hFc, anddid not bind hDII1-hFc.

An alternative BIACORE™ format was used to assess cross-reactivitybetween anti-hDII4 antibody and either hDII1-hFc or hDII3-hFc. Briefly,ligands hDII4-hFc, hDII1-hFc, and hDII3-hFc were each covalently linkedto a CM-5 chip, through amine coupling, to an RU range of about 8,000 to10,000. REGN421, at 300 μg/ml, was injected over the surface of eachchip. REGN421 bound only to hDII4-hFc; no binding was observed to eitherhDII1-hFc or hDII3-hFc. The same result was observed for REGN422 insteadof REGN421.

OCTET™-based binding assays were employed to determine the bindingbetween selected purified anti-hDII4 antibodies and hDII4-hFc,hDII3-hFc, hDII1-hFc, mfDII4-mmh, or mDII4-mFc. Briefly, strepavidinhigh binding FA biosensors (ForteBio, Inc., Menlo Park, Calif.) werefirst incubated with biotin-anti-hK at 5 μg/ml for 10 min at 30° C., toachieve saturation. Biotin-anti-hK-bound biosensors were then incubatedwith antibodies REGN281, REGN421 or REGN422, at 20 μg/ml for 10 min at30° C., to achieve saturation. The antibody-bound biosensors were thenincubated with either hDII4-hFc or hDII3-hFc, hDII1-hFc, or mDII4-mFc,at 200 nM, or mfDII4-mmh at 100 nM, for 10 min at 30° C. Changes in thethickness of the biological layer after each incubation were measured.Human DII4-hFc and mfDII4-mmh bound to anti-hDII4 antibody-boundbiosensors, whereas hDII3-hFc, hDII1-hFc, and mDII4-mFc did not bind toanti-hDII4 antibody-bound biosensors.

Example 9 Effect of Anti-hDII4 Antibody on Tumor Growth

The effect of REGN421 on tumor growth was evaluated on tumors implantedin Severe Combined Immunodeficiency (SCID) mice expressing a humanizedDII4 protein (SCID×hDII4). Briefly, the humanized DII4 mouse was made byreplacing the entire extracellular domain of the mouse DII4 gene withthe corresponding extracellular region of the human DII4 gene (7 kb) inembryonic stem (ES) cells. Homozygous hDII4 mice were generated and bredinto SCID background. Each mouse was then implanted subcutaneously (SC)with 2.5×10⁶ human HT1080 tumor cells. After the tumors were establishedin the mice (˜100-150 mm³ 18 days after implantation), mice weremeasured and treated with hFc, hDII4-Fc or REGN421. A total of 7 micewere divided into three groups. The first group (n=3) was treatedsubcutaneously with hFc at 25 mg/kg; the second group (n=1) was treatedwith hDII4-Fc at 25 mg/kg; and the third group (n=3) was treated withREGN421 at 10 mg/kg. The treatments were repeated every 48 hoursstarting at day 18. In vivo tumor measurements were obtained three daysbefore the initial treatment (day 15), on the same day of each treatment(days 18, 20, and 22), and on day 25. Tumor size was calculated usingthe formula I×w²/2. Results are shown in Table 15. On day 25, mice wereeuthanized and each tumor was removed and measured ex vivo andcalculated (length×width×depth) (Table 16).

In addition, a group of SCID mice expressing endogenous mDII4 (n=2) wasimplanted with tumor cells and treated with hDII4-Fc (25 mg/kg)following the same dosing schedule.

TABLE 15 Tumor Size (mm³) Mouse Treatment Day 15 Day 18 Day 20 Day 22Day 25 SCID hDII4-hFc 162.0 232.8 320.0 336.0 253.1 SCID hDII4-hFc 22.5117.0 117.0 108.0 68.8 SCIDxhDII4 hDII4-hFc 288.0 320.0 352.0 446.0320.0 SCIDxhDII4 hFc 162.0 288.0 320.0 500.0 550.0 SCIDxhDII4 hFc 162.0220.5 352.0 662.0 661.5 SCIDxhDII4 hFc 93.8 135.0 179.6 352.0 726.0SCIDxhDII4 REGN421 144.0 245.0 320.0 162.0 144.0 SCIDxhDII4 REGN421 87.5162.0 153.0 225.0 135.0 SCIDxhDII4 REGN421 144.0 196.0 272.0 162.0 152.5

TABLE 16 Mouse Treatment Tumor Size (mm³) SCID hDII4-hFc 308.0 SCIDhDII4-hFc 105.0 SCIDxhDII4 hDII4-hFc 480.0 SCIDxhDII4 hFc 924.0SCIDxhDII4 hFc 1020.0 SCIDxhDII4 hFc 792.0 SCIDxhDII4 REGN421 168.0SCIDxhDII4 REGN421 84.0 SCIDxhDII4 REGN421 189.0

1. An isolated human antibody or antibody fragment which specificallybinds human delta-like 4 (hDII4), comprising heavy-chain CDR1, CDR2 andCDR3, which comprise the amino acid sequences of SEQ ID NOS:399, 401 and403, respectively, and light-chain CDR1, CDR2 and CDR3, which comprisethe amino acid sequences of SEQ ID NOS:407, 409 and 411, respectively,wherein said antibody or antibody fragment binds an epitope within theN-terminal domain of hDII4.
 2. The human antibody or antibody fragmentof claim 1 being capable of binding dimeric hDII4 with an affinityconstant (K_(D)) of about 75 pM or less, as measured by surface plasmonresonance.
 3. An isolated human antibody fragment which specificallybinds hDII4, comprising a heavy-chain variable region comprising theamino acid sequence of SEQ ID NO:397, and a light-chain variable regioncomprising the amino acid sequence of SEQ ID NO:405, wherein saidantibody or antibody fragment binds an epitope within the N-terminaldomain of hDII4.
 4. The human antibody or antibody fragment of claim 3being capable of binding dimeric hDII4 with a K_(D) of about 75 pM orless, as measured by surface plasmon resonance.
 5. A pharmaceuticalcomposition comprising the antibody or antibody fragment of claim 1 anda pharmaceutically acceptable carrier.
 6. A pharmaceutical compositioncomprising the antibody or antibody fragment of claim 3 and apharmaceutically acceptable carrier.